RAPANA THOMASIANA GROSSE (GASTROPODA) HEMOCYANIN - SPECTROSCOPIC STUDIES OF THE STRUCTURE IN SOLUTION AND THE CONFORMATIONAL STABILITY OF THE NATIVE PROTEIN AND ITS STRUCTURAL SUBUNITS

1. The stability towards pH changes, thermal and chemical (guanidine h ydrochloride) denaturation of the oxy- and apo-forms of the native Rap ana thomasiana haemocyanin and its structural subunits, RHSS1 and RHSS 2, has been investigated using fluorescence and CD spectroscopy. The a ssociation of the subunits into haemocyanin aggregates increases consi derably the melting temperature and the free energy of stabilization i n water, The guanidine hydrochloride denaturation of the aggregated ox ygen-transporting protein depends slightly on the protein concentratio n. The denaturation of the individual subunits is concentration-indepe ndent. Rapana haemocyanin is 5.9-7.5 kJ/mol more stable than the const itutent polypeptide chains. 2. Upon excitation of the native haemocyan in and the subunits at 295 or 280 nm the fluorescence emission is dete rmined by tryptophyl residues 'buried' deeply in the hydrophobic inter ior of the protein globules. This is confirmed by quenching experiment s with acrylamide, caesium and iodide ions. The efficiency of the radi ationless energy transfer between the phenol (donor) and indole (accep tor) fluorophores in the three species, native haemocyanin, RHSS1 and RHSS2, has been determined. An efficient 'interchain' energy transfer between tyrosyl and tryptophyl residues from different polypeptide cha ins occurs in the non-dissociated form of the haemocyanin. 3. The tryp tophan emission of the oxyhaemocyanin, oxy-RHSS1 and oxy-RHSS2 is stro ngly quenched by the copper-dioxygen complex at the active site and th e respective quantum yields of fluorescence of the oxygenated species are 4-7 times lower than those of the ape-forms. Protonated imidazole groups quench the fluorescence of neighbouring excited indole rings, p robably by charge-transfer complex formation.